The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.

PURPOSE: To investigate the effects of INS37217, a synthetic P2Y(2) receptor agonist, on intracellular calcium signaling, electrophysiology, and fluid transport in vitro and on experimentally induced retinal detachment in rat eyes in vivo. METHODS: Freshly isolated monolayers of bovine and human fetal RPE were mounted in Ussing chambers for measurements of cytosolic calcium levels ([Ca(2+)](i)), membrane voltages and resistances, and transepithelial fluid transport. Retinal detachments were experimentally produced in Long-Evans rats by injecting modified phosphate-buffered saline into the subretinal space (SRS). Experimental or vehicle solutions were injected into the vitreous, and the size of blebs in the SRS was scored under masked conditions. RESULTS: Addition of INS37217 to Ringer's solution bathing the apical membrane transiently increased [Ca(2+)](i), altered membrane voltages and resistances and generally produced responses that were similar in magnitude to those of uridine triphosphate (UTP). In fluid transport experiments performed with the capacitance probe technique, INS37217 significantly increased fluid absorption across freshly isolated bovine and fetal human RPE monolayers. All in vitro results were blocked by apical 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which has been shown to block P2Y(2) receptors in the RPE. Intravitreal administration of INS37217, but not UTP, in the rat model of retinal detachment enhanced the removal of SRS fluid and facilitated retinal reattachment when compared with vehicle control. CONCLUSIONS: These findings indicate that INS37217 stimulates the RPE fluid "pump" function in vitro by activating P2Y(2) receptors at the apical membrane. In vivo INS37217 enhances the rates of subretinal fluid reabsorption in experimentally induced retinal detachments in rats and may be therapeutically useful for treating a variety of retinal diseases that result in fluid accumulation in the subretinal space.

The albino chick as a model for studying ocular developmental anomalies, including refractive errors, associated with albinism.

Albinism is associated with a variety of ocular anomalies including refractive errors. The purpose of this study was to investigate the ocular development of an albino chick line. The ocular development of both albino and normally pigmented chicks was monitored using retinoscopy to measure refractive errors and high frequency A-scan ultrasonography to measure axial ocular dimensions. Functional tests included an optokinetic nystagmus paradigm to assess visual acuity, and flash ERGs to assess retinal function. The underlying genetic abnormality was characterized using a gene microarray, PCR and a tyrosinase assay. The ultrastructure of the retinal pigment epithelium (RPE) was examined using transmission electron microscopy. PCR confirmed that the genetic abnormality in this line is a deletion in exon 1 of the tyrosinase gene. Tyrosinase gene expression in isolated RPE cells was minimally detectable, and there was minimal enzyme activity in albino feather bulbs. The albino chicks had pink eyes and their eyes transilluminated, reflecting the lack of melanin in all ocular tissues. All three main components, anterior chamber, crystalline lens and vitreous chamber, showed axial expansion over time in both normal and albino animals, but the anterior chambers of albino chicks were consistently shallower than those of normal chicks, while in contrast, their vitreous chambers were longer. Albino chicks remained relatively myopic, with higher astigmatism than the normally pigmented chicks, even though both groups underwent developmental emmetropization. Albino chicks had reduced visual acuity yet the ERG a- and b-wave components had larger amplitudes and shorter than normal implicit times. Developmental emmetropization occurs in the albino chick but is impaired, likely because of functional abnormalities in the RPE and/or retina as well as optical factors. In very young chicks the underlying genetic mutation may also contribute to refractive error and eye shape abnormalities.

The role of the retinal pigment epithelium in eye growth regulation and myopia: a review.

Myopia is increasing in prevalence world-wide, nearing epidemic proportions in some populations. This has led to expanded research efforts to understand how ocular growth and refractive errors are regulated. Eye growth is sensitive to visual experience, and is altered by both form deprivation and optical defocus. In these cases, the primary targets of growth regulation are the choroidal and scleral layers of the eye that demarcate the boundary of the posterior vitreous chamber. Of significance to this review are observations of local growth modulation that imply that the neural retina itself must be the source of growth-regulating signals. Thus the retinal pigment epithelium (RPE), interposed between the retina and the choroid, is likely to play a critical role in relaying retinal growth signals to the choroid and sclera. This review describes the ion transporters and signal receptors found in the chick RPE and their possible roles in visually driven changes in eye growth. We focus on the effects of four signaling molecules, otherwise implicated in eye growth changes (dopamine, acetylcholine, vasoactive intestinal peptide (VIP), and glucagon), on RPE physiology, including fluid transport. A model for RPE-mediated growth regulation is proposed.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia.

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega. cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R(A)/R(B)) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC(50) of approximately 24 microM (apical) and approximately 30 microM (basal). Apical ATP/UTP (100 microM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R(A)/R(B) by a factor of approximately 3. The addition of adenosine to either side (100 microM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca(2+) concentration (427 +/- 206 nM; 15-25 microM ATP, apical; n = 6). This Ca(2+) increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 microl x cm(-2) x h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca(2+)-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.